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Frequently Asked Questions

Here, you will find the answers to frequently asked questions. In case you cannot find the answer you are looking for, you are cordially invited to direct your inquiry to us via our contact form. You can also contact our local representatives or one of our distributors.

Questions on PEPperPRINT's Technology

Our technology is based on a patented peptide laser printing technology allowing the synthesis of peptides directly on glass slides. For epitope mappings, we translate proteins into 15mer peptides with an overlap of 14 amino acids (corresponds to a shift of 1). This leads to a maximum peptide-peptide overlap and enables a high resolution epitope analysis. A reduction of the peptide-peptide overlap results in an inaccurate outcome of epitope lengths and is therefore not recommended.

We are able to design each array individually and we are flexible in the selection of custom peptide sequences. Therefore, you do not pay per peptide, but per array and, if desired, service. Other providers charge each synthesized peptide. Their peptides are costly pre-synthesized in high amounts and afterwards spotted onto the glass slides. Due to the fact that this pre-synthesis is expensive, other providers offer a lower peptide overlap (correlating with a higher shift) to reduce the number of peptides they have to synthesize.

Our service includes comprehensive data analysis, including epitope prediction and interpretation. A report tailored to your specific project includes all experimental data, images and discussion of the obtained results.

Routinely we print 15mer peptides, but we also provide shorter or longer peptides, up to 20 amino acids in length. For peptides between 16-20 amino acids we need to charge an extra fee per chip. In an optimal setting we could demonstrate a successful synthesis of 25mer peptides: We synthesized an elongated (GS)n linker up to amino acid 17, and added the flag epitope, consisting of 8 amino acids, on top. We could show α-flag antibody binding to these 25mer peptides, meaning that the peptides were synthesized up to amino acid position 25.

For our epitope mapping services we routinely use 15mer peptides and the maximum peptide-peptide overlap of 14 amino acids (equals to a shift of 1). If desired, we can also adjust the peptide lengths and/or the overlap.

Array production takes about 3-4 weeks including quality control steps. Details about the process of peptide array production can be found here.

The spot size is approximately 150x300 µm and the distance between spots is 254 µm (horizontal) and 508 µm (vertical). The peptide concentration per spot is in the lower picomolar range.

We can print 1, 2, 3, 4, 5 or 16 array copies per chip, which can be assayed individually with a matching incubation tray. The number of array copies per chip depends on the number of peptides per array.

  • 1 array per chip with up to 5,500 different peptides in duplicate
  • 2 array copies per chip with up to 2,376 different peptides in duplicate
  • 3 array copies per chip with up to 1,320 different peptides in duplicate
  • 4 array copies per chip with up to 924 different peptides in duplicate
  • 5 array copies per chip with up to 726 different peptides in duplicate
  • 16 array copies per chip with up to 120 different peptides in duplicate

In addition to these peptides, we frame each array with Polio, c-Myc and HA control peptides.

Due to the printing flexibility, spot duplicates can be arranged randomly, beneath or next to each other.

We use a linker consisting of 3 amino acids (2x β-alanine and 1x aspartic acid). The aspartic acid is negatively charged and prevents the unspecific binding of the negatively charged fluorescent dyes to positively charged lysines close to the array surface.

Our assay conditions are adapted to detect epitopes with different degrees of hydrophobicity/hydrophilicity. Even weak epitopes with sub-micromolar affinity can be detected. We assessed the assay sensitivity in an own study. We synthesized Leu-enkephalin peptides and variants on the array and incubated them with an α-Endorphin antibody. We then compared the fluorescent signal intensities with published apparent affinities. We were able to detect antibody binding down to µM binding affinities. This highlights the high sensitivity of our assay. 

We recommend testing your antibodies in Western blot under denaturing conditions. If the antibodies work under these conditions, they likely recognize a linear epitope, which we can map with linear peptides. If you cannot detect bindings in Western blot your antibody might bind to conformational or discontinuous epitopes. To analyze antibodies binding to certain 3D structures, we can print cyclic constrained peptides.

Linear epitopes can be mapped with linear peptides on chip (epitope mapping and epitope substitution scan). For conformational epitope mapping we offer cyclic constrained peptide microarrays that mimic looped structures of epitope.

In addition to the 20 standard amino acids, we offer a variety of different amino acids that can be coupled to the chip. These are:

  • Aminoisobutyric acid
  • Biotin
  • D-Alanine
  • D-Aspartic acid
  • D-Cysteine
  • D-Glutamic acid
  • D-Lysine
  • D-Tryptophan
  • L-Citrulline
  • L-Homocitrulline
  • L-Ornithine
  • L-Tyr(2-Cl-Trt)
  • N-Acetyl-L-lysine
  • N-Methylglycine
  • N-Methyl-L-alanine
  • N-Methyl-L-isoleucine
  • N-Methyl-L-norleucine
  • N-Methyl-L-phenylalanine  
 

    PEPperCHIP® Peptide Microarrays should be stored at 4°C in the dark to ensure a long shelf life. If handled with care, chips are stable for months. If possible, please store them under inert gas atmosphere.

    In theory the arrays can be stripped. However, the stripping efficiency always depends on the nature of the samples and the assays. It may not work in all cases, and the chips may also degrade to some extent. Stripping efficiency has to be checked by microarray scanning, and even then you likely will not know if you removed all primary antibodies, or only the secondary antibody. Since we can print multiple array copies per chip (number of copies depends on the size of the array) we recommend using a new array for each experiment.
    Questions on Quality Assessment and Controls

    PEPperPRINT is ISO 9001:2015 certified. You can find a summary of all quality control steps here.

    Due to the high number of different peptides (up to 70,000 in a discovery format) and the picomolar amount, it is technically impossible to analyze all synthesized peptides in HPLC. Nevertheless, we perform numerous quality tests during production setup: We routinely run double coupling steps to get as close as possible to quantitative coupling yields. After coupling, non-reacted amino groups are capped to avoid the formation of peptides with incomplete sequences. According to HPLC analysis and mass spectrometry with reference peptides, side reactions like aspartimide and β-lactam formation or racemization can be excluded.

    We have developed an own glass slide coating that is optimized for peptide array production and antibody assays, and offers superior signal-to-noise ratios. The arrays are blocked prior to antibody hybridization with either BSA or blocking reagents (like Rockland Blocking Buffer) to prevent unspecific antibody binding.

    Selectivity depends on the specificity of the target antibody. To prevent non-specific binding, the target antibody staining (with first antibody or serum) is performed over night at 4°C.

    We routinely use HA control peptides (YPYDVPDYAG), and our standard arrays include Polio, HA and c-Myc controlpeptides. Other controls are available upon request.
    Questions on Immunoassay Requirements, Handling, and Analysis

    We support you with your optimal array design and project setup. Please contact us to discuss your requirements.

    PEPperCHIP® handling and analysis is straightforward and follows standard immunoassay protocols. To get a first glance about working with PEPperCHIP® Peptide Microarrays, we recommend watching our short tutorial movie. More details are available in our standard immunoassay protocol. Typical lab and material requirements for PEPperCHIP® Peptide Microarray assays are listed below.

    PEPperCHIP® Peptide Microarray analysis can be done in your own lab or by PEPperPRINT experts within a PEPperMAP® Service offer.

    In addition to your PEPperCHIP® Peptide Microarray, the following materials are needed for peptide microarray experiments:

    • PEPperCHIP® Incubation Tray: The tray is adapted to the array layout, fully reusable and allows working with low sample volumes
    • PEPperCHIP® anti-HA control antibodies: Contains labeled α-HA antibodies for staining of the control peptides
    • MAPIX Analyzer: Software solution for peptide microarray image analysis
    • Blocking reagent: Rockland Blocking Buffer


    Material supplied by you (if you decide to perform experiments yourself):

    • Orbital shaker
    • Microarray scanner: The PEPperCHIP® Peptide Microarrays are compatible with most microarray scanners (e.g. GenePix 4000B, 4100B and 4300/4400 Microarray Scanners, Tecan Laser and Power Scanner, Agilent High-Resolution and SureScan Microarray Scanner, Innopsys InnoScan 710 and 900, NimbleGen MS 200 Microarray Scanner, LI-COR Odyssey Imaging System and many more). A detailed scanner list can be downloaded here.
    • Buffers: PBS, Tween20
    • Labeled secondary antibody for staining of primary sample (e.g. mouse α-human antibody). Please note that strongly charged fluorescence dyes can interact with acidic and basic side chains of peptides. We thus recommend the use of neutral dyes or dyes with few charged functional groups (e.g. DyLight680, DyLight800, Cy3, Cy5), as well as pre-incubation with secondary antibodies to screen for background interactions. PEPperPRINT uses Pierce infrared dyes and the LI-COR Odyssey® Infrared Imaging System.
     

     

    All scanners that are compatible with 75mm x 25mm (3in x 1in) microscope slides can be used to scan PEPperCHIP® Peptide Microarrays. We recommend a scanning resolution of 20 μm or higher (10 μm, 5 μm, etc.). Please download our detailed list of compatible scanners here.

    Strongly charged fluorescence dyes can interact with acidic and basic side chains of peptides. We thus recommend the use of neutral dyes or dyes with few charged functional groups (e.g. DyLight680, DyLight800, Cy3, Cy5), as well as pre-incubation with secondary antibodies to screen for background interactions. PEPperPRINT uses Pierce infrared dyes and the LI-COR Odyssey® Infrared Imaging System.
    Questions on PEPperMAP® Services

    During sample analysis, the peptide array is first incubated with the unlabeled primary antibody. Afterwards, the secondary, dye-labeled antibody is added, which binds to the primary antibody. Our service includes comprehensive data analysis, including epitope prediction and interpretation. A report tailored to your specific project and needs includes all experimental data, images and discussion of the obtained results. Considering that every customer project is different we adapt our reports to each project. We provide you with all raw data (Excel format) and a report including the following data:

    • Information about the project
    • Assay conditions
    • Array scans
    • Analysis of the epitope
    • Interpretation of the data
     

      In case the epitope sequence is known, we are able to identify the amino acids that are important for the binding of the antibodyby a substitution scan. In this process, each amino acid of the epitope is replaced one after the other by all remaining 19 L-amino acids (D-amino acids on request). The number of peptides can be calculated by epitope length x number of L-amino acids x number of peptide replicates. For example, a peptide microarray for an epitope with 13 amino acids contains 13 x 20 x 2 = 520 peptides.

      PEPperMAP® Service costs are calculated based on the time and material needed for thorough service. Standard PEPperMAP® Epitope Mapping Services with an antibody or serum sample against a single protein are covered by flat-rate fees that are widely independent of the length of the protein sequence. Please do not hesitate to contact us for more information and nonbinding cost estimations for the intended research project. We guarantee to always provide you with the most cost effective PEPperMAP® Service solutions available.

      The timeline starts with receipt of the antigen sequence, followed by 4 weeks for peptide microarray generation. We usually ask to ship the sample(s) before peptide microarray finalization. Subsequently, the assays will take around 3 weeks followed by another week for data evaluation and quantification. In total, it takes around 8 weeks for the full process until delivery of the report.

      For service to start, we need a purchase order containing the order number and the billing address. Furthermore, we need the peptide list/antigen sequence (FASTA format or Uniprot ID). We will then send you an order confirmation and the layout of the microarray (peptide map) for your approval. After your approval, we will start with the peptide synthesis which will take about 3-4 weeks. During this time, you can send the samples to us. Please inform us as soon as you send the samples and complete the PEPperMAP® Sample Submission Form (containing information about the sample name, storage temperature etc.). Payment can be done via bank transfer (after receiving the invoice) or by credit card.

      PEPperPRINT GmbH
      Tullastrasse 2
      69126 Heidelberg
      Germany

      Please complete and return the PEPperMAP® Sample Submission Form along with your antibody and serum sample(s) to ensure proper handling and storage.

      Most customers send us antibody or serum samples on ice packs using FedEx. If you would like to use dry ice, fill the box half with dry ice. Seal your tubes with parafilm, put them in a plastic bag and put this bag directly in the dry ice. Fill the empty space with Styrofoam to prevent that the dry ice is shaken too much.

      Please describe your goods precisely on the Air Waybill (e.g. human monoclonal antibody, murine serum, synthetic antibody). If you use e.g. “reagent for research” or “protein solution”, FedEx will ask us for more information, which can delay sample shipment.

      For analysis of animal-derived antibodies and sera that are shipped from overseas (e.g. from USA), please complete and return the PEPperMAP® Sample Declaration Form along with your samples to avoid any clearance delay. Animal derived samples are randomly checked by the veterinary office at the airport in Cologne. The veterinary office wants to make sure that no animal diseases or pathogenic agents are introduced into Germany. An inspection by the veterinary office may lead to extra costs.

      To import animal derived samples into Germany, it is necessary that you state in the PEPperMAP® Sample Declaration Form that your samples have been separated from plasma and are highly purified, so that animal disease or other pathogenic agents were killed off or effectively removed. Please insert the sample nature and state how the samples were produced. Please attach this form to your shipment documents to the outside of the box.

      If you have animal serum samples that are not purified, please contact us for further details. Human serum samples and synthetic proteins or antibodies do not require the aforementioned  form.

      First, we need the antigen sequence in FASTA format or the Uniprot ID. We will discuss with you how much serum or purified antibody we need to run the assay services for your specific project.

      The amount of sample needed depends on different factors. One is the incubation volume that depends on the size of the array. Moreover, it depends on whether serum or antibody is used. Last but not least, we test up to three different dilutions for incubation in order to obtain the best results possible. The exact amount of sample needed can therefore only be determined individually for each project.

      Please send us your protein sample/antibody without BSA and without sodium azide. Both additives reduce the labeling efficiency.

      Pre-staining with the secondary antibody is a routine background control.